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組換えヘモペキシンを用いたヘムの細胞内取り込み及び遺伝子発現制御機構の解明

羽田 浩士 東北大学

2020.03.25

概要

【背景】近年、細胞障害等で生じる細胞外のヘムが、細胞内にシグナルを伝達するシ グナル分子として機能し生体応答に寄与することが提唱されている。細胞外のヘムは、ヘモペキシンと結合した状態で存在し、エンドサイトーシスにより細胞内へ取り込ま れることが知られている。エンドソーム又はリソソームまで取り込まれたヘムが、細 胞質に移行し転写因子等へ作用するか不明であり、移行に関わる分子機構についても 明確にはなっていない。そこで本研究では、ヘモペキシンと結合したヘム(ヘモペキ シン結合型ヘム)が、細胞内に取り込まれ、ヘム結合性転写抑制因子である BACH1 に作用するかを検討し、エンドソームに局在するヘムの輸送体である HRG-1 がその取り込みに関与するか調べた。

【方法】哺乳類細胞株由来の組換えヘモペキシンとヘムとを結合させた上で精製することでヘモペキシン結合型ヘムを調製した。これを、マウス肝細胞及びヒトマクロファージ細胞の培養液に添加し、BACH1 への影響を調べた。BACH1 はヘムと直接的に結合することで自身の分解が促進され、ヘム分解酵素の HO-1(遺伝子は Hmox1)の発現抑制が解除される。そのため、BACH1 のタンパク質レベル及び Hmox1 mRNA の発現量を調べることで、細胞外ヘムの取り込みを評価した。細胞外ヘムの細胞内への取り込みについて、エンドサイトーシスに依存するかは、その阻害薬である CPZ を用い、HRG-1 が関与するかは、Hrg-1 遺伝子のノックダウンにより検証した。

【結果】マクロファージ細胞及び肝細胞の両細胞において、ヘモペキシン結合型ヘムを添加したところ Hmox1 遺伝子発現の誘導が見られた。また、その際、肝細胞において、BACH1 のタンパク質レベルが低下していることが分かった。このことから、ヘモペキシン結合型ヘムが細胞内へ取り込まれたことが示された。CPZ でエンドサイトーシスを阻害したマクロファージ細胞では、ヘモペキシン結合型ヘムによる Hmox1遺伝子の発現誘導が抑えられ、ヘモペキシン結合型ヘムの取り込みがエンドサイトーシスに依存することが示唆された。肝細胞において、ヘモペキシン結合型ヘムによる Hmox1 mRNA の発現誘導は、Hrg-1 遺伝子のノックダウンにより、有意に増強されたことから、HRG-1 は細胞外ヘム取り込みを抑制的に機能することが示唆された。

【結論】細胞外のヘモペキシン結合型ヘムが、エンドサイトーシスにより細胞内に取り込まれ、BACH1 の分解を介した HO-1 の発現誘導を担うことが確認された。また、 HRG-1 は細胞外ヘムの取り込みに関与するものの、その機能は、既に報告されているヘムの細胞質への輸送を促進することはなく、むしろ抑制することが示唆された。

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